Nitric oxide (NO) is an important signaling molecule in animals and plants. In mammals, NO is produced from Arg by the enzyme NO synthase. In plants, NO synthesis from Arg using an NO synthase-type enzyme and from nitrite using nitrate reductase has been demonstrated previously. The data presented in this report strongly support the hypothesis that plant tissues also synthesize NO via the nonenzymatic reduction of apoplastic nitrite. As measured by mass spectrometry or an NO-reactive fluorescent probe, Hordeum vulgare (barley) aleurone layers produce NO rapidly when nitrite is added to the medium in which they are incubated. NO production requires an acid apoplast and is accompanied by a loss of nitrite from the medium. Phenolic compounds in the medium can increase the rate of NO production. The possible significance of apoplastic NO production for germinating grain and for plant roots is discussed.

Apoplastic Synthesis of Nitric Oxide by Plant Tissues

An analytical anion-exchange procedure has been developed for the rapid separation of acid-soluble nucleotides (the so-called "free" or tissue nucleotides). It permits assay at low pressures (275-415 kPa; 40-60 psi) in less than 1 h on 10-cm columns of Aminex resins (conventional styrene-type anion exchangers) with alkaline citrate solutions as the eluent. Separation variables have been investigated, to determine optimum conditions for the routine analysis of samples containing tissue nucleotides. Also described here is a simple solvent-extraction procedure for removing HClO4 or CCl3CO2H quantitatively from cell extracts that contain acid-soluble nucleotides: they are removed from aqueous acid solutions with a water-insoluble amine dissolved in a water-immiscible solvent.

An analytical system for rapid separation of tissue nucleotides at low pressures on conventional anion exchangers.

Aluminium (Al) stimulates the efflux of malate from the apices of wheat (Triticum aestivum L.) roots (Delhaize et al. 1993, Plant Physiol. 103, 695–702). The response was five to tenfold higher in Al-tolerant seedlings than Al-sensitive seedlings and the capacity for Al-stimulated malate efflux was found to co-segregate with Al tolerance in a pair of near-isogenic wheat lines differing in Al-tolerance at a single dominant locus. We have investigated this response further using excised root apices. Half-maximal efflux of malate from apices of Al-tolerant seedlings was measured with 30 μM Al in 0.2 mM CaCl2, pH 4.2, while saturating rates of 2.0 nmol·apex−1·h−1 occurred with concentrations above 100 μM Al. The stimulation of malate efflux by Al was accompanied by an increase in K+ efflux which appeared to account for electroneutrality. The greater stimulation of malate efflux from Al-tolerant apices compared to Al-sensitive apices could not be explained by differences in the activities of phosphoenolpyruvate carboxylase or NAD-malate dehydrogenase. Several other polyvalent cations, including gallium, indium and the tridecamer Al13, failed to elicit malate efflux. Aluminium-stimulated efflux of malate was correlated with the measured concentration of total monomeric Al present, and with the predicted concentrations of Al3+ and AlOH2+ ions in the solution. Several antagonists of anion channels inhibited Al-stimulated efflux of malate with the following order of effectiveness: niflumic acid≈NPPB>IAA-94≈A-9-C>ethacrynic acid. Lanthanum, chlorate, perchlorate, zinc and α-cyano-4-hydroxycinnamic acid inhibited malate release by less than 30% at 100 μM while 4,4′-diisothiocyanatostilbene-2,2′-disulphonate (DIDS) had no effect. These results suggest that the Al3+ cation stimulates malate efflux via anion channels in apical cells of Al-tolerant wheat roots.

Characterisation of Al-stimulated efflux of malate from the apices of Al-tolerant wheat roots

SUMMARY
Two types of source contribute photosynthate for grain filling in wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), current photosynthate transferred directly to the grain and photosynthate redistributed from reserve pools in vegetative tissues. Reserve pools provide the substrate needed to maintain transport and the supply of assimilate to grains during the dark period of the diurnal cycle and during the latter part of grain filling, when the photosynthetic apparatus is senescing and the rate of dry matter accumulation of grains exceeds the rate of dry matter accumulation of the total crop. In addition reserve pools provide a means by which the current rates of photosynthate production and of photosynthate use in grain filling are allowed to proceed (at least in part) independently from each other. There is evidence that all photosynthetic organs (leaves, glumes and exposed portion of the peduncle) contain one or more diurnal carbohydrate storage pools. Diurnal storage of sucrose seems to be much more important than the transient storage of starch. There is little evidence for fructan pools serving a role as a net source of carbon during the dark period of the diurnal cycle. However, fructan is the most important longer-term reserve carbohydrate of vegetative tissues. Fructan accumulation occurs mainly in the extended internodes and leaf sheaths and usually terminates within three weeks following anthesis, after which fructan is gradually lost until grain maturity. The accumulation and loss of fructan are greatly influenced by environmental conditions and treatments that alter the longer-term balance between photosynthate production by the plant and photosynthate use in grain filling. Observations are consistent with the view that fructan accumulation in expanded vegetative tissue is not competitive with grain filling, but the fructan pools accept surplus photosynthate during periods of low demand by grains and provide photosynthate during longer-term deficits in current photosynthate production. Even under optimal conditions for photosynthesis it is likely that half or more of the photosynthate in mature grains is temporarily deposited in one or more reserve pools before being transferred to the grain. Neither the efficiency of reserve utilization in grain filling nor its potential variability in different genotypes and environments are known. Also, very little is known about the mechanisms that control the partitioning of photosynthate between the grain and reserve pools and the allocation to different types of reserve pools. Future progress in the understanding of photosynthesis-yield relationships will likely depend to a great extent on improved knowledge of the controls that govern photosynthate deposition and redistribution in the different pools of reserve carbohydrate.

https://nph.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1469-8137.1993.tb03731.x

The role of carbohydrate storage and redistribution in the source-sink relations of wheat and barley during grain filling — a review

The metabolism of one-carbon (C1) units is essential to plants, and plant C1 metabolism has novel features not found in other organisms-plus some enigmas. Despite its centrality, uniqueness, and mystery, plant C1 biochemistry has historically been quite poorly explored, in part because its enzymes and intermediates tend to be labile and low in abundance. Fortunately, the integration of molecular and genetic approaches with biochemical ones is now driving rapid advances in knowledge of plant C1 enzymes and genes. An overview of these advances is presented. There has also been progress in measuring C1 metabolite fluxes and pool sizes, although this remains challenging and there are relatively few data. In the future, combining reverse genetics with flux and pool size determinations should lead to quantitative understanding of how plant C1 pathways function. This is a prerequisite for their rational engineering.

One-carbon metabolism in higher plants

The amino acid composition of endosperm cavity sap and of sieve tube saps from the flag leaf, peduncle, rachis, grain pedicel, and grain were determined for wheat plants just past the mid-half of grain filling. On a mole percent basis, glutamine accounted for almost half of the amino acids in sieve tube sap from the peduncle and ear. Other protein amino acids, plug γ-aminobutyrate, were present in varying, but mostly low (a few mole percent) proportions. The amino acid composition of phloem exudate resembled that of the mature wheat grain. The proportions of amino acids in the endosperm cavity were generally similar to those of the sieve tube sap supplying the grain. Cysteine, however, while virtually absent from sieve tube sap, comprised 1 to 2 mole percent of amino acids in the endosperm cavity, suggesting it is transported in a different form. Also, alanine and, to a lesser extent, glutamate were relatively more prominent in endosperm cavity sap than in the sieve tube sap. Thus, while most amino acids were more concentrated in the sieve tube sap than in the endosperm cavity sap, alanine and glutamate appeared to be moving from the sieve tube to the endosperm cavity in the absence of, or perhaps even against, their concentration gradients.

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Amino Acid Composition Along the Transport Pathway during Grain Filling in Wheat

Lemna perpusilla 6746, grown photoautotrophically at a series of sulfate concentrations ranging from 0.32 to 1,000 μm, was labeled to radioisotopic equilibrium with 35 SO 4 2− . Sulfur-containing compounds were isolated and purified from the colonies. Radioactivity in each compound was a measure of the amount of that compound present in the tissue. The following compounds were identified and quantitated: inorganic sulfate, glutathione, homocyst(e)ine, cyst(e)ine, methionine, S-methylmethionine sulfonium, S-adenosylmethionine, S-adenosylhomocysteine, cystathionine, chloroformsoluble (presumed to be sulfolipid), protein cyst(e)ine, and protein methionine. γ-Glutamylcyst(e)ine, erythro - and threo -thiothreonine, and S-methylcysteine were not detected. No volatile 35 S compounds were formed during plant growth at 1,000 μm sulfate, nor were significant amounts of 35 S compounds excreted into the medium. The amount of each component present in colonies grown over the 3,000-fold range of medium sulfate was relatively constant except for inorganic sulfate. This increased about 30-fold from the lowest to the highest medium sulfate concentration. The total soluble sulfur amino acids increased about 1.5- to 2-fold, due primarily to an increased amount of glutathione. Protein cyst(e)ine and protein methionine were the major organic sulfur compounds in Lemna , and the amounts of these compounds remained virtually constant despite the variation in external sulfate concentration. Procedures for the analysis of S-adenosylmethionine, S-methylmethionine sulfonium, and S-adenosylhomocysteine are presented.

Sulfur-containing Compounds in Lemna perpusilla 6746 Grown at a Range of Sulfate Concentrations

The starchy endosperm (SE) of the developing grain (caryopsis) of barley (Hordeum vulgare L.) cv Himalaya, as well as that of other barley cultivars examined, acidifies during maturation. The major decrease in pH begins with the attainment of maximum grain dry weight, onset of dehydration, and completion of chlorophyll loss. Acidification is correlated with the accumulation of malate and lesser amounts of citrate and lactate, produced and probably secreted by the pericarp/testa/aleurone (PTA). It is accompanied by large concurrent rises in phosphoeno/pyruvate carboxylase and alcohol dehydrogenase (ADH) activity in the PTA. The activity of seven other enzymes of oxaloacetate and pyruvate metabolism was found to fall or rise only slightly during acidification. Sequential changes in relative amount of ADH isozymes were found in both PTA and SE. The PTA maintained a high respiration rate and adenylate energy charge (AEC) throughout acidification, whereas the SE showed a low respiration rate and rising AEC. The data are consistent with the occurrence of hypoxia in the SE. It is suggested that the above enzyme changes are required for the development of a malate/ethanol fermentation (i.e. a mixed metabolism) in the aleurone layer during maturation.

Endosperm Acidification and Related Metabolic Changes in the Developing Barley Grain

The dark respiration rate of discs from fully expanded tobacco leaves (Nicotiana tabacum) increased linearly with decreasing diameter, the relative increase being independent of leaf age The wound respiration responsible for this situation reached a plateau within 15 minutes of excision Metabolite analysis gave evidence for two independent effects, also unrelated to age The first was a forward crossover between phosphoenolpyruvate and pyruvate which was found as early as 1 minute after excision and persisted for up to 40 minutes It was attributed to activation of pyruvate kinase by a changed ionic balance resulting from membrane damage, was accompanied by a reverse crossover between triose phosphates and 3-phosphoglycerate, and was localized in the outer region of the discs The second effect was a rapid rise in hexose monophosphate and ATP levels throughout the discs After 1 to 10 minutes the ATP/ADP ratio rose strongly for at least 3 hours; after 20 to 40 minutes there was net synthesis of adenine nucleotide as ATP These results indicate that extrapolation from leaf discs to intact leaves is highly inadvisable

Rapid Metabolic Changes in the Wounding Response of Leaf Discs following Excision.

The expression of the Adh1 gene (alcohol dehydrogenase, EC 1.1.1.1) was studied in the aleurone layer of barley (Hordeum vulgare cv. Himalaya). Expression increased markedly during grain development at the levels of activity, enzyme protein and mRNA. mRNA content, but not enzyme activity, could be increased further by exogenous abscisic acid (ABA) when isolated, de-embryonated developing grains were pre-treated with gibberellic acid (GA3) or fluridone. In isolated mature aleurone layers incubated with exogenous hormones, ADH mRNA was strongly up-regulated by ABA and down-regulated by GA3 within 6 h. With ABA, this increase in mRNA was followed by an increase in ADH protein and activity, peaking at 18 h. With GA3, the decrease in mRNA was accompanied by simultaneous decreases in protein and activity. In general, GA3 counteracted the effect of ABA and vice versa. In the aleurone of germinating grain, ADH activity decayed in a distal direction from the embryo, consistent with down-regulation by gibberellin(s) diffusing from it. It was concluded that ADH gene expression in the aleurone of the intact grain is regulated by an ABA/gibberellin interaction.

Peter K. Macnicol

Papers

Amino Acid Composition Along the Transport Pathway during Grain Filling in Wheat

Sulfur-containing Compounds in Lemna perpusilla 6746 Grown at a Range of Sulfate Concentrations

Endosperm Acidification and Related Metabolic Changes in the Developing Barley Grain

Rapid Metabolic Changes in the Wounding Response of Leaf Discs following Excision.

Regulation of alcohol dehydrogenase gene expression in barley aleurone by gibberellin and abscisic acid.